LOCUS       sequence-173405-        6516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PV4 gRNA entry vector for CRISPR decoder.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6516)
  AUTHORS   Weinberg BH, Pham NT, Caraballo LD, Lozanoski T, Engel A, Bhatia S, 
            Wong WW
  TITLE     Large-scale design of robust genetic circuits with multiple inputs 
            and outputs for mammalian cells.
  JOURNAL   Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805.
  PUBMED    28346402
REFERENCE   2  (bases 1 to 6516)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805."
COMMENT     Sequence Label: pBW1944_ hU6-PV4-loxP-BbsI-ENTRY-gRNA
FEATURES             Location/Qualifiers
     source          1..6516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        997..1376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1378..1653
                     /label=chicken beta-actin promoter
     intron          1654..2669
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2593..2615
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2677..2696
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(2795..2828)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2858..2867
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2864..2866,2867..2869,2870..3580)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2864..2866,2867..2869,2870..3580)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2909..2930)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3170..3189)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3517..3538
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3581..3634
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3635..4345
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3780..3798)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4048..4067
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4551..4570)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4616..4671
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4670..4689)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5032..5048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5032..5048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5045..5067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5056..5072
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5080..5086,5087..5104,5105..5110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5125..5146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5231..5365
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5385..5519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5422..5441)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5476..5495
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5587..5604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5758..6346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5838..5857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt
     1021 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1081 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1141 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1201 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1261 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1321 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1381 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1441 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc
     1501 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggaa aggtgcggcg
     1561 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1621 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgttgcc ttcgccccgt
     1681 gccccgctcc gcgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1741 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga
     1801 cggctcgttt cttttctgtg gctgcgtgaa agccttaaag ggctccggga gggccctttg
     1861 tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg
     1921 cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct
     1981 ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg
     2041 cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg
     2101 cgcggcggtc gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc
     2161 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg
     2221 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg
     2281 gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc
     2341 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg
     2401 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt
     2461 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     2521 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     2581 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
     2641 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
     2701 ctcatcattt tggcaaagaa ttgattaatt cgagcgaagg cgcgccctga cctcctgcca
     2761 gcaatagtaa gacaacacgc aaagtcgcac gcgtataact tcgtatagca tacattatac
     2821 gaagttatac cggtagtctc gcagcaacgc tcgtgccgcc accatggtga gcaagggcga
     2881 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2941 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     3001 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     3061 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     3121 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     3181 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     3241 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     3301 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     3361 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     3421 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3481 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3541 cgccgccggg atcactctcg gcatggacga gctgtacaag gagggcaggg gaagtcttct
     3601 aacatgcggg gacgtggagg aaaatcccgg cccaatggtg agcaagggcg aggaggataa
     3661 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     3721 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     3781 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     3841 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     3901 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     3961 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     4021 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     4081 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     4141 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     4201 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     4261 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     4321 cggcatggac gagctgtaca agtaagcact agcgtcaggt acgctgaatt cggtaccgag
     4381 ccaactccct ttacaacctc actcaagtcc gttagaggcg gccgcccagg atacatagat
     4441 taccacaact ccgagccctt ccaccgctag cgcactcctc aggtgcaggc tgcctatcag
     4501 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc
     4561 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     4621 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     4681 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     4741 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
     4801 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     4861 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     4921 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     4981 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     5041 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5101 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5161 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     5221 gcaaccatag tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     5281 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     5341 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag
     5401 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5461 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc
     5521 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5581 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5641 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5701 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5761 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5821 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5881 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5941 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6001 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6061 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6121 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6181 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6241 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6301 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6361 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6421 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6481 aatctaaagt atatatgagt aaacttggtc tgacag
//
